Voltage-activated potassium (KV) channels are potassium selective integral membrane proteins formed by the assembly of four homologous subunits. In response to a membrane depolarization, KV channels open allowing K+ to permeate. In some members of KV channels, sustain depolarization leads to inactivation caused by an N terminus gate. In shaker KV channels, the first 20 amino acids at the NH2 terminus of the protein are essential in enabling it to act as a gate. The tip of the NH2 terminus interacts with residues in the intracellular cavity of KV channels, blocking the permeation of K+. By nature of their tetrameric architecture, inactivating KV channels have four N terminus gates and a set of four sites of action in the intracellular cavity. Yet, N-type inactivation is produced by the binding of only one N terminus gate. Is the site of action in the pore specific to the subunit to which the bound N terminus belongs? To study the interactions between the N terminus gate and the site of action we are using thermodynamic cycle between positions in the N-terminus and in the T1 domain. In parallel we are performing molecular dynamic simulations to guide our experimental work. We also study the gating mechanisms of transporters, like the Na/K-ATPase. This enzyme, a member of the P-type family (named for their phosphorylated intermediates), harnesses the energy from the hydrolysis of one ATP to alternately export 3Na ions and import 2K ions against their electrochemical gradients. By performing this active transport, the Na/K pump plays an essential role in the homeostasis of intracellular Na and K that is crucial to sustaining cell excitability, volume, and Na-dependent secondary transport. On the basis of biochemical data accumulated during the decade following its discovery, the Na/K-ATPase was proposed to alternately transport Na and K ions according to a model known as the Post-Albers scheme. As ions are transported through the Na/K pump, they become temporarily occluded within the protein, inaccessible from either side, before being released. By restricting Na/K pumps to only the reversible transitions associated with deocclusion and extracellular release of Na+, it is possible to detect pre-steady state electrical signals accompanying those transitions. The signals arise because Na+ traverse a fraction of the membrane potential as they enter or leave their binding sites deep within the pump. At a fixed membrane potential and external sodium concentration, the populations of pumps with empty binding sites, and those with bound or occluded Na, reach a steady-state distribution. A sudden change of membrane voltage then shifts the Na-binding equilibrium, and initiates a redistribution of the pump populations towards a new steady state. The consequent change in Na-binding-site occupancy causes Na to travel between the extracellular environment and the pump interior. In so doing they generate a current. As the system approaches a new steady distribution, fewer Na move, and the current declines. The electrical signals therefore appear as transient currents. Using the squid giant axon preparation, which exploits axial current delivery to generate very fast membrane voltage steps, we previously identified three phases of relaxation in transient pump currents (Holmgren et al., 2000): fast (comparable to the voltage-jump time course), medium-speed (tm 0.2-0.5 ms), and slow (ts 1-10 ms). We suggested that each phase reflects a distinct Na-binding event (or release, depending on the direction of the voltage change) with its associated conformational transition (occlusion or deocclusion). In other words, the Na/K-ATPase undergoes dynamic rearrangements that open external gates to allow bound Na access to the extracellular environment immediately prior to release. We would like to understand how these gates operate, the precise dynamic relationships between the three events that release individual Na+ from the Na/K-ATPase, the thermodynamic principles that govern these conformational changes, the structural movements underlying these events, as well as the type of structural dynamics associated with them. Recently, genetic mutations in the brain specific Na/K-ATPase (ATP1A3) have been linked to specific human pathologies, like Alternating Hemiplegia of Childhood (AHC), a devastating disease affecting over 120 unrelated children around the world. We have begun to study the functional consequences of some of these mutations, in particular D801N, E815K and G947R.These positions are located within the transmembrane region of the ATP1A3. We hypothesize that because of their location, they might influence ion binding transitions.